Advances in bioanalytical technologies have provided greater and greater amounts of information on the operation of biological processes, how different processes operate together, and how one might influence those processes through the application of external pressures, e.g., through the introduction of outside agents like pharmaceutical compounds, changes in environments, etc. Typically, such analyses rely on the observation of certain highly biologically relevant reactions and processes to monitor their progress, rate, and one's ability to influence them. In some cases, the reactions are employed as tools for identifying the characteristics of other components of the reaction, based upon how the reaction progresses. For example, nucleic acid synthesis reactions have been monitored in order to identify the sequence of the template nucleic acid against which the product is synthesized, by identifying the nucleotides incorporated at each position in that sequence.
In general, these relevant reactions are monitored by looking at populations or ensembles of molecules all reacting together, to provide a monitorable reaction product. These reaction populations or ensembles are typically provided in homogeneous patches or regions on substrates, or in wells of a multiwall substrate.
Such ensemble approaches provide a number of useful properties, including signal availability from larger collections of reactions, averaging of individual molecular reaction variations over the ensemble, and ease of use with conventional measuring tools, e.g., fluidic measurements, optical measurements, or the like. However, some of these traits, while advantageous in one context, are detrimental in others. For example, where ensemble approaches average over the entire population of reactions, one is, as a result, unable to see potentially important events at the individual reaction level. Similarly, by watching only an ensemble reaction, one is often required to artificially start and stop reactions to identify specific events, e.g., through the use of terminating reactants, by limiting necessary reactants, or the like.
For a number of applications, the ability to monitor individual reaction complexes, or single molecule reactions, provides the ability to directly observe a given reaction, as well as those influences upon that reaction. For example, the ability to directly, and in real time, detect the incorporation and identity of nucleotide building blocks into the template directed synthesis of nucleic acids, e.g., DNA and RNA, provides the ability to, by implication, derive the underlying sequence of the particular nucleic acid template. Because individual molecules are being observed, one can continuously observe the synthesis reaction, and thus ‘read out’ extremely long segments of the underlying template sequence.
Systems have been developed that are capable of directly monitoring individual reaction complexes in real time. One such system is the SMRT Sequencing system that employs optically confined reaction volumes coupled to highly sensitive multiplexed fluorescent illumination and detection systems. The result is illumination of very small volumes immediately surrounding an individual immobilized reaction complex in order to excite fluorescent reagents as they are taking part in the reaction of interest. The fluorescent signals are then transmitted in parallel through optical trains to separate signals having different spectral characteristics, e.g., resulting from different reactions, to be directed at sensitive sensor arrays. The position on the sensor array operates to identify the specific reaction that yielded a given signal.
In order to improve the throughput of these systems, one must increase the number of individual reactions that are observed at any given time. Because these reaction complexes are immobilized, this typically involves one or both of increasing the observed area containing the immobilized complexes and/or increasing the density of reaction complexes in a given area. With respect to the latter approach, one often encounters limitations of how densely reaction complexes can be packed on a surface, and still resolve adjacent reactions from each other. In particular, depending upon the nature of the optical system, and the detector used, e.g., the pixel size and count, signals from adjacent reaction complexes, where too closely packed, will be detected as a single unified or otherwise unparsable signal, or will otherwise overlap with each other, such that simple discrimination between two signals becomes more difficult.
The present invention, on the other hand, provides methods systems, and consequently, reaction substrates, that provide the ability to have more densely packed reaction complexes while still permitting the resolution of otherwise unresolvable signals. Further, these same methods are applicable for other applications, including identification and discrimination of individual or multi-reaction signals, and the like.